Benchmarking of real-time, field-deployable whole-genome sequencing of Plasmodium falciparum using Nanopore technology.

Genomic sequencing of pathogens is rapidly becoming a cornerstone of infectious disease surveillance, providing valuable information for disease control programmes. Malaria genomic surveillance has been rapidly expanding with endemic countries increasingly utilizing Nanopore platforms, which are an economical, portable and relatively easy-to-use technology that produces data from samples in real time with a sequencing approach that may uncover blind spots of the genome. Here, we present extensive benchmarking of Nanopore sequencing for whole-genome sequencing of , the most deadly and common malaria parasite in highly endemic areas. We developed an end-to-end workflow for high-quality data and showed that Nanopore performs similarly to gold standard methods and can reveal notoriously difficult-to-access parts of the genome and resolve entire chromosomes. This demonstrates the utility of Nanopore for whole-genome sequencing that could provide greater insights than existing sequencing approaches for use in research and public health. The authors confirm that all supporting data, code and protocols have been provided within the article or through supplementary data files. Whole-genome sequencing (WGS) provides complete information about pathogens that along with epidemiological metadata can enhance control efforts [] to track the spread of pathogens in real time [], the emergence of drug resistance [], responses to control interventions [] and inform vaccine design []. Human malaria, a disease that has plagued humans for thousands of years, is caused by infection with species. It remains one of the world’s most widespread and deadly infectious diseases []. A major roadblock to WGS of malaria parasite isolates has been overcome through methods to enrich trace amounts of parasite DNA from finger prick blood samples contaminated with large amounts of human DNA []. However, generation of WGS has traditionally been restricted to well-equipped laboratories that can maintain large, expensive sequencing platforms as well as advanced analytical pipelines and human resources required for data processing. As Illumina short-read WGS (srWGS) requires extensive human expertise in both library preparation and instrument support, researchers in malaria-endemic countries have generally needed to send samples to large genome centres for sequencing, resulting in significant time delays and loss of data custodianship. However, for genomic surveillance to inform malaria control and elimination in a timely manner, large numbers of genomes with spatially dense sampling and the rapid generation of high-quality data at low cost will be needed, and this is only feasible through sequencing in proximity to endemic areas.